Method of fluorescence analysis comprising evanescent wave excitation and out-of-plane photodetection

ABSTRACT

Fluorescence measuring methods and apparatus use planar optical waveguides to excite fluorescence in the evanescent field of the waveguides and photodetectors to sense the fluorescence produced. Chemically sensitive fluorophores are bound to the evanescent regions of the planar optical waveguides. Substrates support the waveguides. Photodetectors, positioned in the substrates with fields of view normal to the waveguides, detect the fluorescence. Wavelength-selective material coating surfaces of the photodetectors allow fluorescence to be detected while restricting entry of light at the excitation wavelengths. The photodetectors have high aspect ratios for detection of fluorescence generated by the optical waveguides. Preferably, the photodetectors are closely coupled to the fluorescence generated in the evanescent field of the waveguide. In alternative embodiments, lightguides near the waveguides direct fluorescence from the plane of the waveguides for transmission by integrated optical fibers to remote sensors. For chemical detection and analysis, energy is introduced to an edge of the waveguide. The edges are coated to reflect the energizing energy throughout the waveguide. The energy excites the chemically sensitive fluorophore film positioned on the waveguide. Inexpensive photodetectors without expensive photo-multipliers are mounted in substrates beneath the waveguides or at ends of lightguides extending from the substrates for detecting fluorescence.

BACKGROUND OF THE INVENTION

This invention relates to fluorescence sensors for chemical detectionand analysis.

Optical fluorescence sensors are useful in analyzing chemical propertiesof liquid and gas-phase analytes. Existing fluorescence methods andapparatus are based on evanescent wave excitation and in-planefluorescence detection. Those methods and apparatus suffer from lowefficiency of collection of fluorescent light. Effective analysisrequires the use of expensive photomultiplier tubes for adequatefluorescence detection. Drift and other noise effects associated withthe use of photomultiplier tube devices reduce sensor precision andresult in inaccurate analysis. Needs exist for sensors that haveout-of-plane detection and eliminate the need for expensivephotomultiplier tubes.

Evanescent wave excitation allows monitoring chemical reactions within ashort distance of the excitation beam. The sensitivity of chemicalsensors based on evanescent wave excitation is limited by the amount offluorescence coupled to the waveguide. The amount of fluorescencecollected by a photodetector is limited by inefficiencies associatedwith coupling light from the source to the optical waveguide.Additionally, the specificity of a fluorescent sensor is reduced byinterferences resulting from chemical reactions between sensor materialand materials other than the analyte of interest. Needs exist forfluorescent waveguide sensors having increased specificity, sensitivityand precision.

SUMMARY OF THE INVENTION

The present invention provides a highly sensitive optical fluorescencesensor and related method that combine both evanescent wave excitationand out-of-plane detection. The sensor has increased fluorescencecollection efficiencies from a planar surface and functions effectivelyusing inexpensive photodiodes.

The present invention uses an excitation source to excite fluorescencein the evanescent field of a planar optical waveguide. A photodetectorspositioned such that the field of view of the instrument is normal tothe waveguide, detects the fluorescence. The sensor uses low-costphotodiodes for detection, permits realistic chemical sensors to be madefrom thin-layer evanescently excited fluorophores and reduces effectivedevice lengths required in fluorescence detection.

A fluorescence sensor for chemical analysis includes a substrate, awaveguide positioned on the substrate and a chemically sensitivefluorophore film positioned over the waveguide. An excitation sourcepositioned near the waveguide delivers energy to an end facet of thewaveguide. A photodetector is placed proximate to the waveguide. Thephotodetector is positioned such that the field of view of theinstrument is normal to the waveguide. A wavelength-selective materialfilm coats a surface of the photodetector. The coating allowsfluorescence to be detected and restricts light at an excitationwavelength from reaching the photodetector. The photodetector ispositioned a short distance from the waveguide. In one embodiment, thephotodetector is located in a hollow portion of the substrate below anactive region of the waveguide.

The excitation source is located near the waveguide or connected to thewaveguide from a remote location by optical fibers. A prism coupler onan end of the waveguide receives the energy and directs the energy fromthe excitation source to the waveguide. Alternately, a light emittingdiode or a laser diode, is integrated with the waveguide.

The substrate is preferably a glass or polymer substrate. Thephotodetector is preferably a silicon photodiode having high-aspectratio (length/width) and a wavelength-selective interference filter. Inone embodiment, the waveguide is glass disk treated by potassium-ionexchange. A silver film is applied to outer edges of the disk to provideinternal reflection of the energy beam. In a second embodiment, twoclosely spaced channel waveguides form segments of a compositewaveguide. The first segment and the second segment are spaced and laygenerally parallel, creating a flow channel between the segments. Thelower surface of each segment is connected to the substrate. The uppersurface of each segment is brazed or solder connected to thephotodetector.

For detection at remote locations, a collector, such as an opticallightguide, is positioned near the waveguide for collecting fluorescencegenerated by the fluorophores in the waveguide. The collector isconnected to a remote photodetector by an optical fiber. In multiplexedsensors having multiple waveguides, a multiple pronged opticallightguide is used, with each prong positioned near a single waveguidefor collecting fluorescence generated by the single waveguide. Where thewaveguide is segmented, the collector is an optical fiber arraypositioned near the channel for collecting fluorescence generated by thewaveguide.

The method for fluorescence analysis using evanescent wave excitationand out-of-plane photodetection involves preparing a waveguide on asubstrate and introducing energy to an end of the waveguide. Energy isintroduced directly by coupling the excitation source to the waveguideor indirectly through a prism positioned on an end of the waveguide. Theenergy produces fluorescence in a chemically-sensitive fluorophore boundto an evanescent region of the waveguide. The fluorescence is detectedusing a photodetector having a field of view normal to the waveguide.For precise and accurate detection, the photodetector allowsfluorescence to pass but restricts light at an excitation wavelengthfrom reaching the photodetector. For detection at remote locations, thefluorescence is collected by a collector and transmitted through anoptical fiber to a remote photodetector.

The present invention can be incorporated in automobiles, airplanes andagricultural equipment. The sensors are useful in chemical processcontrol and in environmental applications. The present invention, whenemployed by automobile manufacturers, provides for precise andinexpensive emissions monitoring and hydrogen vapor monitoring. Firmsengaged in distributed chemical analysis can use the present inventionwith existing chemically sensitive instruments to obtain accuratechemical analysis.

These and further and other objects and features of the invention areapparent in the disclosure, which includes the above and ongoing writtenspecification, with the claims and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a fluorescent sensor having asubstrate, a waveguide positioned on the substrate, a prism coupler, achemically sensitive fluorophore film positioned on the waveguide, and aphotodetector proximate to the waveguide for detecting fluorescenceescaping the waveguide.

FIG. 2 is a schematic side illustration of an optical fluorescencesensor showing removal of substrate material to allow positioning of thephotodetector close to the waveguide.

FIG. 3 schematically shows an optical lightguide positioned near awaveguide for collecting fluorescence and delivering the fluorescence toa remote photodetector.

FIG. 4 schematically shows a multiplexed fluorescence sensor havingmultiple waveguides and a multiple pronged lightguide for collectingfluorescence and for delivering the fluorescence to a photodetectorarray.

FIG. 5 schematically shows a fluorescence sensor having a glass diskoptical waveguide.

FIG. 6 illustrates how the glass disk optical waveguide of FIG. 5operates as an optical resonator as the excitation beam reflects inwardoff side edges of the disk.

FIG. 7 is a schematic illustration of a microflow-injection fluorescencesensor having a double ridge waveguide, a flow channel between theridges of the waveguide and a photodetector positioned above the flowchannel.

FIG. 8 is a schematic illustration of a microflow-injection fluorescencesensor having a double ridge waveguide, a flow channel between theridges of the waveguide and an optical fiber array positioned above theflow channel for collecting fluorescence and for delivering thefluorescence to remote photodetectors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIGS. 1-8, a fluorescence sensor 1 uses evanescent waveexcitation and out-of-plane photodetection to provide precise, sensitiveand inexpensive chemical analysis. As shown in FIGS. 1 and 2, a planaroptical waveguide 3 is positioned on a substrate 5. A chemicallysensitivefluorophore film 7 is deposited over the top surface 9 of thewaveguide 3. Energy from an excitation source 11 is directed to an end13 of the optical waveguide 3. As shown in FIG. 1, one embodiment of thepresent invention uses a prism coupler 15 positioned on an end 13 of thewaveguide3 to direct the energy onto the waveguide 3. The energyproduces fluorescence in the chemically sensitive fluorophore film 7bound to the evanescent region of the waveguide 3. A photodetector 17detects the fluorescence generated.

FIGS. 1 and 2 show one embodiment of the present invention wherein thephotodetector 17 is closely coupled to the waveguide 3. The substrate 5has a hollow portion 19 below the active region of the waveguide 3. Thewaveguide is comprised or two or more dielectric layers which may bedeposited on a substrate 5 or on a buffer layer over the photodetector17.A region of the waveguide may be overlaid by a third dielectrichaving higher refractive index than the guiding layer of the waveguideto increase interaction with the fluorescent source. A photodetector 17,suchas a silicon photodiode, is positioned in the hollow portion 19 ofthe substrate 5. The proximity of the photodetector 17 to thefluorescence source allows a large fraction of the fluorescence to becaptured by the photodetector 17. Preferably, the photodetector 17 ispositioned such thatthe field of view of the instrument is normal to thewaveguide 3.

In preferred embodiments, the photodetector surface is coated by awavelength-selective material that allows fluorescence to be detectedbut restricts light at the excitation wavelength from reaching thephotodetector 17.

FIGS. 3 and 4 show preferred embodiments of the present invention havingphotodetectors 27 positioned at remote locations. An optical waveguide29 is situated on a substrate 31 or on the surface of the lightguide 33.The waveguide 29 can be a dye doped polymer waveguide carryingfluorophores. Excitation energy or light is delivered to an end of thewaveguide 29, generating fluorescence. A collector 33, such as alightpipe or lightguide, is positioned in the vicinity of the waveguide29. The collector 33 allows a considerable portion of fluorescencegenerated by the excited fluorophore to be brought into the plane of thewaveguide 29 for collection and transmission. Optical fiber 35 extendsbetween the collector 33 and the remote photodetector 27 and carries thefluorescence to the photodetector 27.

FIG. 4 shows a multiplexed fluorimeter having multiple waveguides 29 anda multiple pronged collector 33. Light is introduced to a common end 37of the waveguides 29. The fluorescence generated in each waveguide 29 iscollected by a corresponding prong of the collector 33. The collector33, via optical fibers 35, delivers the fluorescence generated by eachwaveguide 29 to a remote photodetector array 39. This embodiment isparticularly useful for solvent identification.

FIG. 5 shows one embodiment of the present invention having adisk-shaped waveguide 43. The waveguide 43 is a glass disk that istreated by potassium-ion exchange to prepare an optical waveguide on thesurface. Thewaveguide 43 is positioned on a substrate 45, such as aglass substrate. A chemically sensitive fluorophore film 47 is appliedto the upper surface 49 of the waveguide 43. Preferably, the edges 51 ofthe disk waveguide 43 are silvered such that light propagating in thewaveguide 43 undergoes multiple reflections. FIG. 6 shows the path of alight beam 53 as the beam53 is reflected off the walls 55 of the diskwaveguide 43. Multiple internal reflection of the excitation light beams53 leads to efficient energy transfer to the fluorophore film 47 locatedin the evanescent fieldof the waveguide 43.

A photodetector 57, such as a photodiode, detects the fluorescence. FIG.5 shows a preferred embodiment wherein the photodetector 57 ispositioned inthe substrate 45 directly beneath the disk waveguide 45. Inan alternative embodiment, a collector, such as an optical lightguide,in proximity to the waveguide 43 collects the fluorescence and transmitsthe fluorescence via an optical fiber located in the plane of thewaveguide 43. Light can be directly introduced to the waveguide 43 by anoptical fiber 59.

In all embodiments of the present invention, the waveguide 43 ispositionedon a substrate 45. Preferably, the optical waveguide 43 isprepared by laser ablation, sol-gel deposition, polymer-solventdeposition or any other coating process. By depositing a waveguide 43having a thickness equal to or greater than 50 microns, excitationsources, such as laser diodes and light-emitting diodes, can be directlycoupled to an end of thewaveguide 43.

FIGS. 7 and 8 show microflow-injection system embodiments of the presentinvention. Fluorescence is generated in a flow channel 61 formed as thespace between two ridge waveguides 63. The segmented waveguides 63 arepositioned on a substrate 65. A high aspect ratio photodetector 67extendsover the channel 61 and is connected to upper surfaces 71 of thewaveguide segments 63. In preferred embodiments, the waveguide segments63 and the photodetector 67 are connected by adhesive joints 73 orbrazement. Preferably, the photodetector 67 is positioned such that thefield of viewof the instrument is normal to the upper surfaces 71 of thewaveguide segments 63. The embodiments shown in FIGS. 7 and 8 areadvantageous in coupling fluorescence generated by an optical waveguide63 integrated witha light emitting diode or laser diode to aphotodetector 67 for monitoring fluorescence associated with chemicalchanges. The use of high aspect ratio wavelength-selectivephotodetectors greatly enhances the collection of fluorescence generatedin the evanescent field of an optical fiber. FIG. 7 shows one embodimentof the present invention incorporating a silicon photodetector 67 havinghigh aspect ratio and a wavelength-selective interference filter. FIG. 8shows another embodiment wherein the photodetector 67 includes anoptical fiber array 75 positionedabove the flow channel 61.

While the invention has been described with reference to specificembodiments, modifications and variations of the invention may beconstructed without departing from the scope of the invention, which isdefined in the following claims.

We claim:
 1. A sensor apparatus for detecting fluorescence comprising a substrate, a waveguide comprising two or more dielectric layers positioned on the substrate, a chemically sensitive fluorophore film positioned over the waveguide, an excitation source positioned near the waveguide for delivering energy to an end facet of the waveguide, and a photodetector placed proximate to the waveguide and positioned such that a field of view of the photodetector is normal to the waveguide wherein the substrate has a hollow portion below an active region of the waveguide, and wherein the photodetector is positioned in the hollow portion of the substrate.
 2. The apparatus of claim 1, further comprising a wavelength-selective material coating positioned over a surface of the photodetector that allows fluorescence to be detected and restricts light at an excitation wavelength from reaching the photodetector.
 3. The apparatus of claim 1, further comprising a prism coupler positioned on an end of the waveguide for directing energy from the excitation source to the waveguide.
 4. The apparatus of claim 1, wherein the substrate is a glass substrate and the photodetector is a silicon photodiode.
 5. The apparatus of claim 1, wherein the waveguide further comprises a glass disk treated by potassium-ion exchange and a silver film applied to outer edges of the disk.
 6. The apparatus of claim 5, wherein the substrate is a glass substrate and the photodetector is a silicon photodiode.
 7. The apparatus of claim 1, further comprising an optical fiber connected to the excitation source and to the waveguide for integrating the excitation source to the waveguide, and wherein the waveguide contains a section overlaid by high refractive index material to enhance interaction with the fluorophore.
 8. A sensor apparatus for detecting fluorescence comprising a substrate, a waveguide comprising two or more dielectric layers positioned on the substrate, a chemically sensitive fluorophore film positioned over the waveguide, an excitation source positioned near the waveguide for delivering energy to an end facet of the waveguide, and a photodetector placed proximate to the waveguide and positioned such that a field of view of the photodetector is normal to the waveguide, wherein the waveguide has a first segment and a second segment lying generally parallel to the first segment, thereby creating a channel between the segments of the waveguide.
 9. The apparatus of claim 8, wherein each waveguide segment has an upper surface and a lower surface, the lower surface connected to the substrate, and wherein the photodetector is connected to the upper surfaces of the waveguide segments.
 10. The apparatus of claim 9, wherein the photodetector and the segments of the waveguide are connected by adhesive joints.
 11. The apparatus of claim 9, wherein the photodetector comprises a silicon photodiode having high-aspect ratio and a wavelength-selective interference filter.
 12. The apparatus of claim 11, wherein the waveguide is a polymer waveguide, wherein the substrate is a glass substrate.
 13. The apparatus of claim 9, wherein the excitation source is selected from a group consisting of a light emitting diode and a laser diode, and wherein the excitation source is integrated with the waveguide.
 14. The apparatus of claim 1 wherein the waveguide has a thickness of at least 50 microns.
 15. A sensor apparatus for detection at remote locations comprising a substrate, at least one waveguide positioned on the substrate, the waveguide carrying fluorophores, an excitation source positioned near the waveguide for delivering energy to an end of the waveguide, a collector positioned near the waveguide for collecting fluorescence generated by the fluorophores in the waveguide, a remote photodetector and an optical fiber connected to the photodetector and to the collector, wherein the collector is an optical lightguide, and wherein multiple waveguides are positioned on the substrate, and wherein the lightguide has multiple prongs, with each prong positioned near a single waveguide for collecting fluorescence generated by the single waveguide.
 16. A sensor apparatus for detection at remote locations comprising a substrate, at least one waveguide positioned on the substrate, the waveguide carrying fluorophores, an excitation source positioned near the waveguide for delivering energy to an end of the waveguide, a collector positioned near the waveguide for collecting fluorescence generated by the fluorophores in the waveguide, a remote photodetector and an optical fiber connected to the photodetector and to the collector, wherein the waveguide is a dye doped polymer waveguide, deposited on the surface of the collector.
 17. A sensor apparatus for detection at remote locations comprising a substrate, at least one waveguide positioned on the substrate, the waveguide carrying fluorophores, an excitation source positioned near the waveguide for delivering energy to an end of the waveguide, a collector positioned near the waveguide for collecting fluorescence generated by the fluorophores in the waveguide, a remote photodetector and an optical fiber connected to the photodetector and to the collector, wherein the waveguide has a first segment and a second segment lying generally parallel to the first segment, thereby creating a channel between the segments of the waveguide, and wherein the collector is an optical fiber array positioned near the channel for collecting fluorescence generated by the waveguide.
 18. The apparatus of claim 1, wherein the waveguide is comprised of at least two or more dielectric layers, and wherein the excitation source is directly coupled to the waveguide, and wherein the waveguide is deposited on a low-refractive index buffer layer, deposited on the photodetector. 